Topical phage therapy in a mouse model of Cutibacterium acnes-induced acne-like lesions

Acne vulgaris is a common neutrophil-driven inflammatory skin disorder in which Cutibacterium acnes (C. acnes) is known to play a key role. For decades, antibiotics have been widely employed to treat acne vulgaris, inevitably resulting in increased bacterial antibiotic resistance. Phage therapy is a promising strategy to combat the growing challenge of antibiotic-resistant bacteria, utilizing viruses that specifically lyse bacteria. Herein, we explore the feasibility of phage therapy against C. acnes. Eight novel phages, isolated in our laboratory, and commonly used antibiotics eradicate 100% of clinically isolated C. acnes strains. Topical phage therapy in a C. acnes-induced acne-like lesions mouse model affords significantly superior clinical and histological scores. Moreover, the decrease in inflammatory response was reflected by the reduced expression of chemokine CXCL2, neutrophil infiltration, and other inflammatory cytokines when compared with the infected-untreated group. Overall, these findings indicate the potential of phage therapy for acne vulgaris as an additional tool to conventional antibiotics.

. Line 133-136; this section regarding the SLST type can be moved to legends of Supp Fig. 11. Line 138-139: "Moreover, the four phage-resistant strains were susceptible to all five tested antibiotics" The author can expand the discussion on this phenomenon. Does that mean the phage is only useful to antibiotic-resistant bacteria but not the sensitive ones? Phage-resistant bacteria? Perhaps this is related to the patient's status where the bacteria isolated?
12. Line 144: Bacteria strain #27 are antibiotics (Abx)-sensitive bacteria (according to Supp Table 2). Therefore routine Abx treatment is already useful to alleviate the pathology. Phage therapy is raising interest because of the "Abx-resistant bacteria". The authors should also test the phage cream on mice with Abx-resistant bacteria infection. 13. Line 145: How did the author determine "high in vitro efficacy"??? as the author only state "S" or "R" in the results.
14. Line 146: "with no difference over a cocktail of phages" How was this conclusion drawn from Supp Table 2? 15. Line 147-148: Data? Did the author also formulate the rest of the phages?
16. Line 147-148: Also, when formulating a cream-based product, especially containing a biologicalactive component, we usually consider their stability as well as microbial content upon storage. (different cream base may affect the viability of the phage) Although this is not the aim of this study, in my opinion, the author may consider discussing it. 17. Line 165 and throughout the manuscript: 1.4*10^4 should be 1.4×10^4. 18. Line 162, 169-171: "(for more details see "Assessment of Phage Clinical Efficacy" section in Materials and Methods and Fig. 7)" this statement should not be presented in the result section. 19. Fig 4: Statistics should also compare between days (e.g. before the treatment) in the same group. 20. Try to simplify Fig 3.  28. Line 210: average area "of the lesion"… sounds better; Also, please add the standard error. 29. Figure 5 and Line 203-204: "…neutrophils surrounded by macrophages and rare lymphocytes,…" None of these cells can be seen in the figure (in such a low power field). The author can only suggest cellular infiltration from the image presented in Fig 5. The author should provide images at a higher power field.
30. Figure 5.:Any reason for combining the score together? How come the sample size differs in the combined score from others? 31. Figure 5: Neutrophil staining has to be done to confirm that the infiltrated neutrophils are indeed resolved by the treatment. (More evidence is needed to support the author's theory!) 32. Line 219: "Three mice were sacrificed during the experiment" Are these mice from those used in Figure 5 or are these extra mice?
33. Figure 6E: What does this figure imply? 34. Table 2: What is the phage used here? 35. Line 265-266; 268-269; 270-275: Because the bacteria were sensitive to either phage or Abx, so this is foreseeable that the bacteria is sensitive to combinational therapy. Therefore, the conclusion drawn by the authors may not be correct (also in the abstract).
36. Line 265-266: In vivo or in vitro model using C. acnes strain resistant to both phages and Abx should be used to confirm this statement. 37. Line 309: " Table XX"??? 38. Figure 6: A phage control (uninfected mice using the cream) should also be investigated in the study to exclude the baseline level of immune activation by the phage 39. At last, the author should also expand their discussion on phage resistance, as this is another problem when using phage therapy. (In this study there are also phage-resistant bacteria) 40. The discussion would flow more smoothly if it were more integrated. The current discussion section sounds more like a result section to me. 41 Fig 6D). Also, where are the data from day 7?   Figure 3: Did image capturing and pathology study also take place on day 8? (as shown in Supp Table  3) 63. Line 805-806: Again, the macrophage and neutrophils cannot be distinguished from the figure.
64. Figure 6A-C: The group name should be added to the figure.
65. Figure 6A-C: Gating strategies should be added in Supp Figure   66. Figure Table 3: The author should take all the pictures from the same angle, light source, background,  etc. For example, images on day 8 from control animals #15 and #16 are totally different than the rest of  the picture, which made us difficult to interpret the data. Other examples such as Day 7 from treated  animal #1; Day 8 from treated animal #16; as well as others!   69. Supp Table 3: Also, the author should provide a scale bar (e.g. ruler with a scale bar next to the lesion site) when taking the picture. This work shows that use of phages in a mouse model of Cutibacterium acne brings a benefit, although not a notably large benefit.
The work is well designed and executed, although its subsequent write-up is less attractive.
The manuscript needs to be carefully and thoroughly edited to achieve correct scientific English. There are numerous mistakes in spelling, punctuation, grammar, and incorrect use of capital letters. The sentence in line 450 no verb.
The term 'sacrifice" which generally means 'slaughtering an animal or person as an offering to a deity" is an unacceptable euphemism for "kill"; "sacrifice" should be replaced throughout with a less euphemistic term such as kill or euthanase.
The main issue with this work seems to be around the animal model. The paper would be improved by: (i) Discussion justifying the suitability of the model, and the route of delivery of the bacteria. Why is it injected, and not, for example, applied to scarified skin? Given that the model required two injections of a relatively large volume of bacteria at high concentration, is the model really representative of what happens in acne?
(ii) Improved description of how the phage was applied. Neither Materials and Methods nor Figure 3 gives enough detail about how the phage was applied. Was the gel simply smeared over the skin lesion?
If so, what volume of gel? Was any intervention used to keep the gel on the lesion and stop the mice from rubbing it off? If not, for how long was the gel in place on each lesion? Dear Dr. Hayleah Pickford, Editor of Nature Communications, Please find our point-to-point answers to the reviewers' comments on our manuscript, termed "Toward phage therapy for acne vulgaris: Topical application in a mouse model of Cutibacterium acnes-induced acne-like lesions" NCOMMS-22-20 by Rimon et al. We want to thank both the reviewers and you for the constructive comments that improved our manuscript significantly.Accordingly , as requested,we revised the manuscript and added experiments supporting our conclusions, including immunofluorescence and ELISA. In addition, we sent the manuscriptagain for language editing (see attached certificate) and hope that the language is now much .better

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): In this study, the authors isolated new C. acnes phages and have found that they are useful in killing C. acnes in vitro. By applying the phage-cream formulation to C. acnes-infected mice, the inflammatory reaction was significantly alleviated. And this may be due to the reduction of neutrophil infiltration. However, some descriptions of results and conclusions appear too superficial and general, and thereby generate, as far as I can see, wrong statements.
 We thank the reviewer for these comments. Accordingly, we elaborated the results thoroughly and altered the general conclusions to state our findings accurately (see details below).
First, a general comment: There are errors in the use of English throughout that require attention and editing. Throughout the manuscript, some rearrangement of the content and inclusion of some more linking sentences might help to improve cohesiveness.
 We apologize for these oversights. Given that we are non-native English speakers, we sent the manuscript for professional editing. However, the editing was unsatisfactory. Hence, we sent the current version of the manuscript for English editing to Wiley Editing Services (https://wileyeditingservices.com/en/ ,see attached certificate). We truly hope that the revised manuscript meets the journal's standards.
My comments that follow are more general, but follow the manuscript seriatim, focusing on the results, underlying assumptions, and conclusions, which, to my mind, need more critical interpretation.  Based on the reviewer's comment, we provided annotation data in Supplemental Table 2 3. As all these phages were isolated from Israel (I assumed), the authors may expand their discussion by comparing their phage to other phages discovered.
 We thank the author for raising this interesting point. Accordingly, this has been elaborated in lines 390-405 4. Line 118 and throughout the manuscript: Length and diameter: the standard error should be added. Which virus family do all these phages belong to (according to TEM results)?
 We added standard diviations throughout the manuscript. We added Supplemental Fig. S1 with an example of transmission electron microscopy (TEM) images for all 8 phages, along with the measured length and capsid diameter for each phage in Table 2. Given the high genetic similarity, we did not observe a significant difference in TEM. According to sequence and TEM findings, isolated bacteriophages belong to the Siphoviridae family.

Line 121-122: (8)
 This was a typographical error, which has been deleted in the revised manuscript.  We are unsure if we comprehensively understand this comment. We did include the results in Table S2 in our manuscript, referred to in line 126. Nevertheless, in the revised manuscript, we included plaque images of each phage for each bacteria ( Fig  S2A), indicating that phage-resistant strains are indeed sensitive to clindamycin ( 7. Line 127-130 and Figure 2D: How come the control group dropped compared to the CM group?
 Currently, we are unable to explain this finding; however, we can confirm that this phenomenon is reproducible within this strain, in which clindamycin was shown to inhibit autolysis 1,2 . It should be noted that this is not a general phenotype but a strain-specific effect. It needs to be determined whether clindamycin causes direct or indirect inhibition of autolysin, an enzyme known to be involved in autolysis.
We elaborate on this point in the discussion in lines 355-359 in the revised manuscript.  We moved this section to the legend of Supp. Fig S3   11. Line 138-139: "Moreover, the four phage-resistant strains were susceptible to all five tested antibiotics" The author can expand the discussion on this phenomenon. Does that mean the phage is only useful to antibiotic-resistant bacteria but not the sensitive ones? Phage-resistant bacteria? Perhaps this is related to the patient's status where the bacteria isolated?
 We detected no clear correlation between antibiotic resistance/sensitivity to phage resistance/sensitivity (Supp Fig S2E). In our collection of 36 strains, we found 21 strains sensitive to both and 4 and 11 sensitive only to antibiotics or phage therapy, respectively. Indeed, we did not detect any strain resistant to both, but we cannot exclude the possibility of such mutant existence that it was not found, perhaps, due to the small size of the present collection. Regarding the question posed by the reviewer, in our opinion, the decision to utilize phages against antibiotic-sensitive bacteria or only in cases of antibiotic resistance should be at the treating clinician's discretion. We elaborate on this point in the discussion in lines 385-389, 439-442 in the revised manuscript.
12. Line 144: Bacteria strain #27 are antibiotics (Abx)-sensitive bacteria (according to Supp Table 2). Therefore routine Abx treatment is already useful to alleviate the pathology. Phage therapy is raising interest because of the "Abx-resistant bacteria". The authors should also test the phage cream on mice with Abx-resistant bacteria infection.
 In the present study, we examined the efficacy of topical phage therapy in vivo.
Establishing C. acnes-induced lesions in mice can be challenging. Mice naturally resist this bacterium and are spontaneously cured following lesion induction without treatment. Furthermore, our antibiotic-resistant strains were less virulent than sensitive ones for yet unknown reasons. Eventually, after screening several isolated strains and identifying a more virulent strain, and with the addition of human sebum, we finally succeeded in inducing lesions that lasted a few weeks. This virulent strain was antibiotic-sensitive. Given that the objective of the present study was to demonstrate the concept of phage therapy, we did not insist on finding a virulent antibioticresistant bacteria. Moreover, based on our findings and those reported previously, there appears to be no clear correlation between antibiotic resistance and phage susceptibility 3,4 . As described in lines 414-429 in the revised manuscript. This point was further elaborated in the discussion section of the revised manuscript. Nevertheless, if the reviewers find this assay essential for establishing the validity of the manuscript, we will conduct this experiment; however, considerable time will be needed to identify the best antibiotic-resistant strain for lesion establishment, as described above.
13. Line 145: How did the author determine "high in vitro efficacy"??? as the author only state "S" or "R" in the results 14. Line 146: "with no difference over a cocktail of " How was this conclusion drawn from Supp   This statement was deleted.
19. Fig 4: Statistics should also compare between days (e.g. before the treatment) in the same group.
 In the revised manuscript, statistical analysis between days was incorporated in Fig.  3 (Formerly Fig. 4).

Try to simplify Fig 3.
 We reduced the redundant wording and elements. Additionally, we rearranged and combined Fig. 3 with Fig. 4 (now numbered Fig. 3). We believe these changes will adequately simplify  We added all time points to Fig. 3 in the revised manuscript.
Is there any reason for using the combined score ( Figure 4K)?
 To grade the lesion, we used multidimensional parameters, and the score is an integration of these parameters. We believe that a single parameter cannot accurately describe the lesions.  The score of the histological analysis was based on the interpretation of an independent expert animal pathologist. We established a quantitative scale based on this interpretation, allowing the comparison between the groups, added to Supplemental Figure S7; see below.
Pathologist report Score WNR (Within normal range) 1 The subcutaneous fat and skeletal muscle (panniculus carnosus) have a nodular to diffuse subcutaneous infiltration with neutrophils and macrophages.

2
The subcutaneous fat and skeletal muscle (panniculus carnosus) have a nodular to diffuse subcutaneous infiltration with neutrophils and macrophages. There is central necrosis within the infiltrate. 28. Line 210: average area "of the lesion"… sounds better; Also, please add the standard error.
 We agree with the reviewer's comment and corrected it accordingly. We also added standard deviation throughout the manuscript.
29. Figure 5 and 30. Figure 5: Any reason for combining the score together?
 We believe that score integration adds simplicity and allows clinicians to decide and follow treatments based on the lesion condition. Nevertheless, we also provide each score parameter (Fig 4).
How come the sample size differs in the combined score from others?
 The histological score refers only to mice that underwent histological evaluation, while the clinical score refers to all mice employed in the study. In the revised manuscript, we only presented scores for mice that underwent histological assessment.
31. Figure 5: Neutrophil staining has to be done to confirm that the infiltrated neutrophils are indeed resolved by the treatment. (More evidence is needed to support the author's theory!)  In accordance with the reviewer's comment, we added immunofluorescence staining for Ly6G+ cells to quantify neutrophils in each group (new figure 5 A-C).
The experimental details were added in the results in lines 301-306 and in the methods in lines 559-567 of the rvised manuscript. All images can be found in the BioStudies data repository (https://www.ebi.ac.uk/biostudies/studies/S-BSST889).
32. Line 219: "Three mice were sacrificed during the experiment" Are these mice from those used in Figure 5 or are these extra mice?
 Please note that these are additional mice. We refer to these mice in lines 538-541 and Table 4 in the revised manuscript.
 This figure implies the significance of the differences between each animal group compared to the uninfected group; this is shown as a graph of p-values. We revised the figure and respective legend, hopefully clarifying the implied meaning.
34. Table 2: What is the phage used here?
 We apologize for the poor clarity regarding the same. By "phage", we meant to imply any tested phage, given the absence of differences between them. This has been clarified in Table 3 (previous Table 2), along with an added note. 35. Line 265-266; 268-269; 270-275: Because the bacteria were sensitive to either phage or Abx, so this is foreseeable that the bacteria is sensitive to combinational therapy. Therefore, the conclusion drawn by the authors may not be correct (also in the abstract).
 As mentioned above, phages do not specifically infect antibiotic-resistant bacteria. However, considering treatment, phage therapy is unessential when antibiotics exhibit sufficient efficacy. In cases when antibiotic-resistant strains are involved, phage therapy should be considered, and even then, probably combined with antibiotics. We do not anticipate the replacement of antibiotic therapy with phage therapy, but phages could be employed as an additional antibacterial tool.
In the revised manuscript, we clarified this important point in lines 347-349 in the revised manuscript. Other lines described by the reviwer were removed from the revised manuscript.

Line 265-266:
In vivo or in vitro model using C. acnes strain resistant to both phages and Abx should be used to confirm this statement.
 In vitro stimulation of antibiotic-resistant strain is described in Fig. 2. We addressed the issue for the in vivo model in comment #12 37. Line 309: " Table XX"???  This has been corrected in the revised manuscript.
38. Figure 6: A phage control (uninfected mice using the cream) should also be investigated in the study to exclude the baseline level of immune activation by the phage  We added this data as Fig 6D-E in the revised manuscript.
39. At last, the author should also expand their discussion on phage resistance, as this is another problem when using phage therapy. (In this study there are also phageresistant bacteria)  We agree with the reviewer that phage resistance is one of the most important issues concerning phage therapy. In general and very briefly, in contrast to antibiotics, when resistance emerges against phages, several steps can be employed to overcome it. First, phages can be evolutionarily mutated and selected, as in nature, to infect the resistant bacteria. Second, new phages can be isolated against resistant bacteria. Finally, phages can be suitably engineered. Moreover, although bacteria have numerous resistant systems, constantly being discovered every other day, we ultimately selected phages that efficiently infect the bacteria, regardless of these systems. We elaborated on these points in the discussion, in lines 372-388 in the revised manuscript 40. The discussion would flow more smoothly if it were more integrated. The current discussion section sounds more like a result section to me.
 We revised the discussion to afford better flow and readability. 41. Was an ELISA ever done to look at serum and cell-released levels of IL-1B?
This may provide information about the general state of the mice.  We thank the reviewer for this comment. Accordingly, ELISA was performed to measure serum levels of excreted IL1-ß in each mouse group. This data is presented in Fig. 6G in the revised manuscript and discussed in lines 330-331 in the result section and in line 602 in the methods section of the revised manuscript.
 For clarity, we used the term "skin swabs" throughout the revised manuscript.

Line 432: 5 mM
 This error has been corrected.
44. Regarding sample collection and phage isolation: Was IRB approval obtained to collect the sample from patients? How was the sample collected? What are the characteristics of these patients? Are they Abx responsive? Did the saliva also collect from acnes pts? And more details are needed.
 In accordance with the reviewer's suggestion, we added the requested data in a new table ( Table 1 in the revised manuscript) and the Materials and Methods section, i.e., lines 444-473 of the revised manuscript. We also added a section in the discussion regarding the connection between patients' bacteria and phage isolation line 372-378 of the revised manuscript 45. When were the uninfected mice euthanized? How were they treated?
 Uninfected mice were euthanized after 3 days (before phage application) and on days 5 and 7 (after phage application). These were administered saline, along with sebum application. We included these details in Table 4   The primers were designed using Primer-Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast). This was added to the revised manuscript in lines 555-556. 49. Line 520: "RT-PCR For RNA isolation": RT-PCR was not used for RNA isolation  We apologize for this error, which has been corrected in the revised manuscript.  Yes, this statement indicates the approval for animal experimentation. We rewrote it to improve clarity and provided another ethical approval for the clinical specimen isolation.
55. Table S4: Were these primers newly designed for the study? If so, please provide more details for each of them.
 The primers were obtained from previous reports, and references were added accordingly in Table S6. 56. Line 450-453: But there are five samples on day 5 (Fig 6D). Also, where are the data from day 7?
 We apologize for this error. The day number was corrected throughout the manuscript. Figure Legends: 57. Table 2: which phage?

Figures and
 By "phage," we meant to imply each phage, given the lack of differences between them. See also our answer to comment 34. This point was clarified in Table 3 (previously 2) and the respective legend.
58. Table 2 legend: "Some isolates were verified as non-C. acnes and therefore some numbers are missing" what does this means?
 Sample numbers were determined during appropriate sampling. However, some bacterial isolates were not identified as C. acnes by 16S analysis and were removed from the study. Nevertheless, these were included in Table S4 in the revised manuscript.
59. Figure 1: The figure is difficult to see. 62. Figure 3: Did image capturing and pathology study also take place on day 8? (as shown in Supp Table 3)  We apologize for this error. The numbering in Supplemental Table 3 was erroneous and was rectified.
63. Line 805-806: Again, the macrophage and neutrophils cannot be distinguished from the figure.
 This has been rectified in the figure and respective legend.
64. Figure 6A-C: The group name should be added to the figure.
 As requested, this was rectified, and the order was altered to match the sequence in the text.
65. Figure 6A-C: Gating strategies should be added in Supp Figure    The gating strategies were added as Supplemental Fig. S8B in the revised manuscript.
66. Figure 6F: I can't see from the figure which group was used as a normalizer to calculate relative expression? (The value of the normalizer seems to differ in each group?)  We corrected Figure 6F; the uninfected group was used as a normalizer.  Figure legends were revised throughout the manuscript, with additional details supplied. We hope that these revisions improve their clarity and implications. Table 3: The author should take all the pictures from the same angle, light source, background, etc. For example, images on day 8 from control animals #15 and #16 are totally different than the rest of the picture, which made us difficult to interpret the data. Other examples such as Day 7 from treated animal #1; Day 8 from treated animal #16; as well as others!  Images were obtained from the same location and in the same room with similar lighting using the same camera. However, owing to the movement of mice, it was impossible to achieve identical angles and backgrounds. Unfortunately, improving the images at this point would warrant the additional euthanization of 34 new animals, and we do not believe that can be justified. However, we added a scale bar to the image to aid in better interpretation. Table 3: Also, the author should provide a scale bar (e.g. ruler with a scale bar next to the lesion site) when taking the picture.

Supp
 As mentioned in the previous comment, we added a scale bar for each image using the measured size of the lesions. Other minor points for improvement: 74. An abstract should be typed as a single paragraph.
 In the revised manuscript, the abstract is presented as a single paragraph.  "Phylogenetic". This was rectified in the manuscript.
Line 299: type in italics  As requested, the text is presented in italics.
 This error has been rectified in the revised manuscript.
Reviewer #2 (Remarks to the Author): This work shows that use of phages in a mouse model of Cutibacterium acne brings a benefit, although not a notably large benefit. The work is well designed and executed, although its subsequent write-up is less attractive. The manuscript needs to be carefully and thoroughly edited to achieve correct scientific English. There are numerous mistakes in spelling, punctuation, grammar, and incorrect use of capital letters. The sentence in line 450 no verb.


Given that our team comprises non-native English speakers, we had the manuscript undergo professional English language editing, which was insufficient. The revised manuscript was once again edited by a native English editor, Wiley Editing Services (https://wileyeditingservices.com/en/), who also supplied a certificate for it. We truly hope that it is much better now.
The term 'sacrifice" which generally means 'slaughtering an animal or person as an offering to a deity" is an unacceptable euphemism for "kill"; "sacrifice" should be replaced throughout with a less euphemistic term such as kill or euthanase.


We replaced the term "sacrifice" with "euthanize" throughout the manuscript.
The main issue with this work seems to be around the animal model. The paper would be improved by: (i) Discussion justifying the suitability of the model, and the route of delivery of the bacteria. Why is it injected, and not, for example, applied to scarified skin? Given that the model required two injections of a relatively large volume of bacteria at high concentration, is the model really representative of what happens in acne?


Herein, we aimed to simulate acne vulgaris as feasible. Acne is a human disease and does not have clinical correlates in animals. Therefore, establishing an animal model simulating acne pathogenicity can be challenging. We thoroughly reviewed the literature and selected a mouse model described by Kolar et al. 8 , which mimics human disease better than other reported animal models 8,9 . This model involved the intradermal administration of bacteria, similar to other accepted acne animal models, as well as the application of artificial sebum to best simulate the environment of the human hair follicle. Thus, in order to induce the lesions, we had to apply high concentrations of bacteria. Note, though, that at the endpoint, when assessing CFU counts, the number of bacteria was much lower. Nevertheless, our major goal in this work was to demonstrate the feasibility of treating skin infections induced by C. acnes using phages. Despite the limitations, this goal was achieved and opened the way for further experiments toward acne vulgaris phage-based treatments. We discussed this in lines 414-429 of the revised manuscript.
(ii) Improved description of how the phage was applied. Neither Materials and Methods nor Figure 3 gives enough detail about how the phage was applied. Was the gel simply smeared over the skin lesion? If so, what volume of gel? Was any intervention used to keep the gel on the lesion and stop the mice from rubbing it off? If not, for how long was the gel in place on each lesion?


We added all requested data on the phage application in lines 534-541 in the revised manuscript. Briefly, 0.5 ml of Carbopol gel containing 10 FD3 phage was applied daily to each lesion. We could not prevent the mice from rubbing it off; however, based on our observation, the gel was still fully present after 3 h and partially detectable after 6 h post-administration. We aimed to establish a treatment protocol that may be relevant in clinical settings. A unique feature of phages is auto-dosing, as phages undergo replication on their target bacterium.
Please find our point-to-point answers to the reviewers' comments on our manuscript, entitled "Toward phage therapy for acne vulgaris: Topical application in a mouse model of Cutibacterium acnes-induced acne-like lesions" NCOMMS-22-20 by Rimon et al.
We want to thank the reviewer for the constructive comments that improved our manuscript significantly, and we agree with all the comments provided and revised the manuscript accordingly.
The major changes we made were the addition of the control of the saline injection, showing that Cutibacterium acnes-induced acne-like lesions resulted from bacterial infection and not from injection per se. In addition, we revised the skin-penetration phage assay Please see below our point-to-point revisions (in blue):

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): 1. A major problem with the animal experiment is that the authors did not present the results of the control group which is essential (such as in figure 4) to data interpretation. As intradermal injection may also induce some histological changes, lacking the control group made us impossible to understand whether the histological changes were indeed caused by bacterial colonization and not due to injections.
 We thank the reviewer for this comment and agree that testing the effect of the injection itself is an important control. To this end, we injected mice with saline and performed all the described assays for this control. For the histology, Fig. 4 was revised accordingly, and we also referred to this is in the text (lines 285-287 in the results section and lines 442-443 in the discussion).
2. Another problem: "…as the control group here was also treated with phage gel" (this is only a treatment-control, which excludes side effects of the phage), what about mice being injected but not being treated with phage gels? (this should be the control group) This group should be presented in all experiments.
 As described above in comment #1 for the histology, the saline injected control group was not treated with phages and was added to each relevant assay. It appears now in Figs 3-6 and the relevant text.
3. The author mentioned that the gel was still fully present after 3 h and partially detectable after 6 h post-administration; does it mean it was not absorbed well into the skin? Did the author perform any tests to determine that the phage was indeed being absorbed? If the gel were still present on the skin, did the author remove them after a period of time?
 We assessed phage presence inside the lesion after disinfecting the outer layer of the lesion (Fig. 3B). This experiment demonstrated that phages had been absorbed into the lesion.
To further test whether the phages' penetration into the skin was not due to the injection tract, we performed an improved ex-vivo assay where phages in Carbopol gel were applied on uninjured non, infected, nor injected skin, followed by an assessment of phage quantification in a segregated compartment. We found that phages indeed penetrate the skin, as shown in Fig S5. For more details, see the next comment.
Regarding gel removal, the gel was not removed because it was applied as a simulation of clinical application, where the gel is left to absorb without intervention. This is now clarified in line 564 of the revised manuscript. Fig S5, please provide the uncropped agar images. Also, if the phages were dropped on the skin fragment, how can the author make sure that the phages droplet on the skin can not be washed out by the medium? Also, details regarding this experiment may need to be clearly explained, such as the thickness and sizes of the skin? Is there any reference supporting this experimental design? There are multiple experiments and devices used to detect drug penetration, is this experiment any better than those?

In
 We thank the reviewer for this comment. We supply further evidence for the ability of phages to penetrate the skin. In Kumar et. al from 2012 1 , it was shown that E. coli targeting phages penetrated a full-thickness mice back skin. We used a similar model to show that our anti-C. acnes phages are also able to penetrate. To this end, we used the model described by Rohrschneider et al 2 where skin pieces were placed in donor compartments of trans-wells. A solution of phages mixed with the water soluble Allora Red dye was applied on the uninjured skin, followed by assessment at several time points of the dye and titer of phages in the recipient compartments (Fig. S5). This experiment demonstrated that the phages penetrate the skin while the dye, representing the solvent, does not These experiments are described in Fig. S5, results (Lines 192-199), discussion (Lines 434-436), and methods (Lines 637-648) sections of the revised manuscript.
5. The use of English was much improved in the revised manuscript, but some mistakes were still spotted and the author may wish to check carefully again before their final manuscript can be published. Also, the author please check again regarding their data presentation, such as the labeling of intercepts.
 Although we used two English editing services, we found and corrected some more typos and errors, including in some axis labels in the figures. We apologize and hope the language of the whole manuscript is acceptable now. 6. S1 legends: typo error of "x40K"?
 This typo was corrected according to the target journal's guidelines. These parameters are also described in lines 161-174 of the revised manuscript.
8. S4 legends: What is the composition of the control tube?
 The composition of the control tube is the phage in Wilkins medium. We clarified this in the legend of Fig. S4: "PFU was calculated from a gel tube and a matching control tube containing a phage in Wilkins medium." This was also clarified in line 162 of the revised manuscript.
9. Line 86: the word "phage" does not need to be in italics